Genome Engineering Workshop: Julia Joung, CRISPR Screens

hi good afternoon everybody so today I'd like to give an overview about how to perform and design crisper screens and this is an overview about what we've published a few years ago the details on how to perform a crisper screen in the nature protocols paper from 2017 so today I would like to just give you the overall highlights of what's important when designing and performing a crisper screen as well as any updates we've had to the protocol since the publication forward to these screens are very powerful tools for studying any genes that lead to a particular phenotype of interest traditionally this was done with Drosophila and what people traditionally did was they with chemically mutagenize flies and then after growing the Flies up picking out which ones had interesting phenotypes that they wanted to focus on then they would go back and figure out which genetic perturbations in the five interest led to that phenotype so basically for genetic screens are very powerful tools for mapping specific genetic perturbations to phenotype now the poem with the Drosophila screens is that going back to figuring figure out which perturbations led to the phenotype of interest was very difficult to do and with the advent of shrna and CRISPR catheline screens this became a lot easier so to give you a brief overview as the genetic screens work by base pair complimentary preparing to the mRNA of interest so what you can do is you can introduce a pool of thousands of SH RNA sequences into a into a pool of cells then to figure out which genes led to your phenotype of interest you would just sequence the shrna and you can tell by because shRNA compliment the MRNA of interest which gene was perturbed to get to that phenotype of interest now the problem with srna's is that even though they did lead to a lot of interesting discoveries especially in the early days shRNA screens were hampered by the fact that shrnas are often nonspecific so you could get hits where it was producing the phenotype of interest just because the shRNA has a particular off target with CRISPR what we have is the CRISPR caste knockout as well as activation screens with CRISPR compared to shrnas CRISPR catheline had fewer off targets which means that the screen that you are getting had better signal-to-noise ratio just to give a brief overview again the way catheline knockout works is that you can cleave the mammalian genome at a particular designated site guided by us sgrna and once you cut that site they there is most likely and anything she Jerry immediate repair which means that there there's an insertion or deletion and from there you are likely to get a frameshift in your protein sequence and that leads to depletion of your target mRNA and protein with Castile activation so this casting is basically a catalytically inactive cast line what it does is it becomes a programmable DNA binder today here I will cover the casa activation but of course all the principles that apply to get the Cassadine activation also applies to repression and other epigenetic modifiers for screening in our lab the Dec has an activator that we worked on is called Sam that has an activator whether consists of is three different activation domains have been built on to the dead cast nine the BP 60 for PC c5 and hsf one activation domains together this forms an activator complex that can very robustly activate target genes when you position this deck as an activator at the promoter and after positioning the this leads to mRNA of regulation so together the CRISPR cal sign and dick has nine binders have become very useful in discovering a series of different phenotypes of interests so I will just name a few of these things more and more every year there's there's a lot more different phenotypes that people have been interested in screening but just to list a few examples Kristopher kokal science screens have been very useful for drug and toxin resistance discovering genus sensuality and more recently people have used them to study the viral host factors as well as immunotherapy now going beyond the coding genes you can also engineer CRISPR libraries to target non-coding elements and that has been a recent interest because now you can study what these non-coding elements do at large scale and basically you can do a CRISPR catheline screen for any biological process with a screen of a phenotype so what do I mean by a screen both phenotype there are three main types of screening selection that is possible so for example if the blue cell here has a perturbation that leads to your screening phenotype then in the positive selection screen which typically gives you the best signal-to-noise ratio in the positive selection screen after you apply the selection pressure you get more of these blue perturbations and riched at the end of the screen and so you can say that your your target cell is positively enriched in the negative selection screen the the exact opposite where the blue perturbation is no longer found at the end of your screen typically negative selection screens are a bit more difficult to perform and require slightly higher coverage because you are measuring a decrease in signal and the last one print perhaps the most versatile one is the marker gene selection where you care about one or more genes that lead to your particular phenotype and after you apply the screening pressure you can apply a series of different methods whether it's by sorting a reporter gene or doing singles RNA seek you can select for your perturbation of interest at the end the marker gene selection screens typically are even though they're more versatile they're a bit more difficult to perform because generally the the type the ways that you would go about selecting for this particular perturbation of interest are not as scalable as for example a positive or negative selection screen so that's something to keep in mind so to give a very broad overview of how the steps are involved in crisper casting screening first you start with choosing or designing a library and then you package this library into lentivirus for delivery and into your pool of cells of interest and then you apply a screening selection to your pool cells where you then enrich or deplete the cells with a perturbation of interest and then at the end of the screen you isolate the genomic DNA and based on the guide distribution in this genomic DNA you can then select candidate genes for validation so now I'll go into detail about each of the steps so to start out the library construction part depending on whether or not you are picking an existing library we're designing your own this can take anywhere from a few weeks to many more weeks so starting with the easy parts the ready-made libraries on a gene so right now there's a lot of different libraries on a gene I will talk about what our lab has put out so far and also mentioned some of the new ones I recommend using so first the knockout libraries it from our lab we have the Gecko libraries which many of you probably have heard of the Gecko library is common either a 1 factor system where the cast line and the guide RNA are on the same vector or a 2 vector system where we have the calcite and guide library separate typically people would choose a 2 vector system because it's easy to make a cell line with a cath line and then just introduce a library at low mo I afterwards after building the cell line and the second one that we've put out is the activation library the activation library because the deck has my activator from our lab is mo has multiple components this comes in a 2 vector or 3 vector system in the Gecko library there are six guide RNAs per gene in the library and then in the Sam library there's three guide RNAs targeting the promoter regions of each gene now moving beyond the Gecko and Sam libraries more recently there's been a number of libraries put out by the GP P which I would recommend looking at if you don't already have the Gecko library I think from the knockout the most recent one might be the Brunello library and then from activation I think they have a newer version out as well so those would be worth looking into if you're just starting out because those libraries have better on target and off target scoring systems more up-to-date on target off target organ systems now if you're looking at a marker gene screen for example and you don't want to have to deal with all the genes in a genome skill library where you can do is you can design that targeted library based on an existing library so in the protocol we have a script for isolating the guide RNA of interest based on the list target genes that you want to want to isolate and so again typically you would do this if you want to screen as a smaller scale and I'll talk about the scale the screen afterwards but a screen typically a genome so screen will require hundreds of millions of cells so if you're doing as marker genes screen for example and you don't want to deal with that many cells you would want to scale down your library to only genes that you care about in many instances this could be genes that are expressed on the cell surface or kinase this for example that you are interested in and so the last one is a de novo library design we also provide the script in the protocol for designing a devote the Nova library this could be very useful if for example you are designing a what we call a bashing screen where you are interested in a hundred kilobases of genomic DNA non non-coding region surrounding a gene of interest and you want to identify any interesting regulatory elements in the area then you probably want to design a custom library from scratch because there's no existing library that targets a region of interest so just briefly the way the library design works is that you have your genome and interest genomic region of interest then you look at all the guys that are in that region and then we will select guys based on minimizing off target activity and maximizing on target activity as well as a few other filter criteria like GC content and homo polymer stretches these two criteria make the guides easier to synthesize by existing companies and we will also include multiple guys per target to offset any potential off target activity and include alternating guides as control so moving on we then if you are designing starting out with a custom library we provide the detail steps on how to scale up the Porcia but briefly you would synthesize I'll go pool using for example twist or custom array and then you would PCR amplify these all the goes then you would restrict restriction digest your plasma backbone of interest and then Gibson in your library into the plasma backbone then before you put this into e e coli there's an isopropanol precipitation stuff to purify the DNA and throughout this cloning stuff the difference between colonial library then this versus a single plasma is that throughout this process we maintain representation of the screen by scaling up the reactions accordingly and we have a table for how to scale this up in the paper so then moving on before we start the screen it's very important to do a quality control step where we will sequence the library that you've combed and make sure that all the guys that you think is there should is actually there and the library is not too skewed so we do this using next-generation sequencing where we will do a PCR to amplify the guides and then we sequence at a depth of 100 reads per guide and then based on the guided distribution if you followed if while you're preparing the library you've followed all the steps that we listed and you're all ago manufacturers very good then you should have about 70% of the guys should be perfectly matching and with lower than 0.5% undetected and this few ratio here refers to the top 10% versus the bottom 10% of your reads what that ratio is so how skewed your library is so then after NGS verification then we proceed to package the live library into lentivirus and the reason why for screens we always do lentiviral who are almost always to lentiviral delivery is that the lentivirus integrates into the genome of your cells so if you're doing a positive selection screen or a negative selection screen where you're measuring measure cell growth or death then the number of copies of your guide RNA will be representative of how many cells you have from that particular gut and in the protocol we include both a left a lipo effect amine version and a P I've urgent and so you can pick and choose depending on what you want to do in the protocol we list like affecting me mm but now we're using lap effect I mean 3,000 and I've posted this the details of this particular protocol are CRISPR Google forum if anyone is interested basically the difference is that lipo effectively 3000 is much more efficient at less than half the cost and so it was a no-brainer to switch from 2,000 to 3,000 for us and if you are interested in non time dividing cells you can also use a V for delivery okay then the trans that once you have the library then then we move into putting this into your pool of cells of interest now before every single screen is very important to calculate the lentiviral tighter because what you want to make sure is that it is most likely that every single cell you only have one copy of the guide RNA if you start integrating multiple guide RNAs into one cell that you are confounding your the effects of that particular guide RNA so in this before screening we always do a lengthy bio tighter step and what does this is we will either swim factor or mix depending on your cell type of interest the infection is more efficient than mixing but at the same time not all cells can handle being spun for two hours and so we provide both messes and what you do is you put different amounts of lentivirus onto yourself and then after selecting for three days using the whatever drug corresponds to your library then you can calculate the multiplicity of infection which is a ratio of the cells that have survived your selection versus the cells that did not survive selection and for the sgrna library you want to make sure that you use the mo of less than 0.3 this ensures that you don't have to start out with a gazillion cells and you at the same time you try to minimize a number of multiple guy number of cells that have multiple guide RNAs in your screen and then from from here after calculating the lentiviral tiger you will scale up accordingly using the same exact method that you use to do the Tai Turing in the first place so how do you scale the transaction so as I alluded to previously a screen is a lot of cells for the transduction of a stream part by up of the screen if you have a hundred thousand guide RNAs in your screen and you want a coverage of 500 cells per sgrna which we recommend and you have an mo I have 0.3 this means that you need to start with 167 million cells and during your screen after the selection you only need to maintain a hundred thousand guide RNAs x 500 cells per guy in RNA which is about 50 million cells so every single time you passage of cells you need to make sure that you retain 50 million cells to make sure that you maintain the coverage of your screen so to give you an example for a knockout stream what you would do is if you are using a two vector system you would introduce a caste 9 and as an mo I of less than 0.7 for a caste line it's the last important that you use an mo I have less than 0.3 because you don't really care if you one cell has multiple copies of caste line and then again you transducer guide RNA library at my F point less than point three and then four knockout screens we typically start the screen seven days from the start of the library show instruction because this is where the in Dells will saturate after seven days and then during the screen you can maintain representation at 500,000 cells per guide RNA and four screens it's very important to do multiple by reps just like any experiment and so you will start out with usually two by ups I would say if this is your first time setting up the screen because you're not sure if going to work and then for very noisy screen you can increase this up to four by reps an activation screen is very similar to a knockout screen where you put in all the components first and then you put in the library but the difference is that for an activation screen you can start the screen at as soon as four days after library transaction because these cells have already been selected for the library and for activation you don't need to wait for you know the activations to saturate it usually is pretty quick so once some of the main considerations during screening selection is that this this really changes for every screen how you do the screen selection but there are a couple of main points that I want to get across because these are things that apply to every single screen the setup of every single screen so first you want to pick parameters that maximize the difference between experimental and control conditions if you are lucky there's probably a few genes that serve as good positive and they give the controls that you can use to set this this number whether it's the drug dose or other conditions for drug dosing since a lot of people do this typically we will screen it the ic50 of the drug because this is like if there is selection pressure but it's not so strong that you're killing off most of yourself and so for drug selections we typically use ic50 again very important throughout the screen maintain the coverage of 500 cells per your guide RNA this will reduce the noise in your screen and if this is your first time setting up a screen and you don't really know at what time you should be harvesting the cells just collect multiple time points you can always resuspend the cells freeze some down through in the minus eighty and it's not a lot of work but if you have these time points and at the end of your sequencing if you realize that you need to go back to the other tone points it's very easy to just fall another vial of cell pellet so it's very important to do this especially if you don't know what time scale you're going to be looking at and during screening it's very important that we keep all the experimental protocols consistent so if you are doing the well tighter one way do the exact same thing when you're scaling up the screen use the same a lot of Fes throughout your screen because a screen is already very noisy so you want to minimize noise as much as possible by keeping your technical procedure very consistent okay so then moving on to harvesting and analyzing the screen in the protocol we describe how to use trigger but at this point Ruger is fairly outdated most people in our lab actually use magic to analyze our screens that I would recommend looking into using magic for analyzing your screens but rigger magic and any other screening analysis program are very similar in that they follow like a similar trajectory where you first first normalize the experimental guide counts to control for positive and negative selection screens usually for drug dosing for example you will have a drug treated and DMSO treated control for flow screens typically you will have the hybin versus a lobe in and the lobe in is your control so you do this normalization to account for any initial starting skew of the library and then you rank your guides and then both of these methods will use the so take each stream look at all the guide rankings for that particular gene and then calculate how enriched each particular gene is relative to random chance and then looking at your by reps you would take either the average enrichment value or the overlap depending and this depends on the person either one both of both methods should look first produce very similar results in this case so at the end of the screen is very very important that you validate your candidate genes a screen only produces a ranked list of candidate genes and you have no idea if well unless all some of them have been characterized in literature you really don't know for sure if that particular gene actually produce a phenotype that you are interested in so for every screen it's very important to go back and buy the guide RNA so for validation the process is extremely similar to doing a normal screen except now you're replacing the library with a single guide RNA targeting a single gene of interest so you repeat this entire process and one second I think [Music] okay but now we're missing the yeah it's just late onset I guess we could do this okay so for a knockout screen this means that after you do the put in the guide RNA you extract the genomic DNA and then you verify that there is in doubt and your screening phenotype occurs once you put in the individual guide and because not all in dulse will deplete the protein it's also very important than you do a Western blot to verify that your protein of interest is depleted and then the protocol we list out a very fast way to do a two-round PCR for evaluating in dose so for the activation screen we want to do then is verify the activation or repression you want to verify that your RNA is increased or decreased as you would expect in the protocol we provide this whole home brew protocol for a rapid extraction of RNA as well as reverse transcription it's very similar to the cells to CT protocols by thermo except thermal cells this product this kits for I think a couple thousand dollars per plate and RS cost maybe 1% of that so highly recommend looking into this basically what had the way it works is that you make your lysis buffer you throw the lysis buffer on to your cells PayPal but down a couple times and then you can transfer the cell lysate directly from the plate into a reverse transcription reaction and then do your reverse transcription which takes about an hour then you can directly do TaqMan qPCR on that reverse transcribed reaction so it's very quick and if you're doing a lot of rtq pcr this is a very quick way and cheap way to do it so again you want to verify your screening phenotype and also do a Western blot to make sure that your protein was actually over expressed as you expect so just to highlight again the most important consideration is during a screen is like I repeated several times you want to maintain coverage throughout the screen at 5 at least 500 cells per guide RNA in your screen now for noisier screens or for instance for negative selection screens you might want to increase this number to 1000 or 2000 and it's also very important to include controls for a screen library the controls are for example the non turning guides in the library itself so you can look at how the non targeting guides in your library changes before and after selection to figure out how much noise was in your library and known as tRNAs that well as journeys that target known genes are sure the fact your phenotype should be used to select your screening parameters and you should always include the negative control by rap in your screen which is the condition that did not have your screening selection and again throughout the screen remain consistent use the same protocols for screening and validation I just have my acknowledgment slides so if anyone has questions I can take questions now yeah so this is actually a very ancient photograph of our lab most of the people in this picture aren't here anymore but the people who really helped me set up this protocol taught me all the initial stuff about screening are in this picture and so I like to acknowledge them um for example Neville as a lot of people know I was also a screening person and he helped write part of the protocol as well thank you [Applause]

Channel: Broad Institute

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Length: 30min 1sec (1801 seconds)

Published: Mon Jul 29 2019